Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields

ABSTRACT

The present invention relates to methods and devices for separating particles according to size. More specifically, the present invention relates to a microfluidic method and device for the separation of particles according to size using an array comprising a network of gaps, wherein the field flux from each gap divides unequally into subsequent gaps. In one embodiment, the array comprises an ordered array of obstacles in a microfluidic channel, in which the obstacle array is asymmetric with respect to the direction of an applied field.

This application is a continuation of U.S. patent application Ser. No.10/693,091, filed Oct. 23, 2003, now U.S. Pat. No. 7,150,812, whichclaims priority to provisional application Ser. No. 60/420,756, filedOct. 23, 2002, the contents of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and devices for separatingparticles according to size. More specifically, the present inventionrelates to a microfluidic method and device for the separation ofparticles according to size.

BACKGROUND

Separation by size or mass is a fundamental analytical and preparativetechnique in biology, medicine, chemistry, and industry. Conventionalmethods include gel electrophoresis, field-flow fractionation,sedimentation and size exclusion chromatography [J. C. Giddings, UnifiedSeparation Science (Wiley, New York, 1991)]. Gel electrophoresisutilizes an electric field to drive charged molecules to be separatedthrough a gel medium, which serves as a sieving matrix. The moleculesare initially loaded at one end of a gel matrix, and are separated intocomponent zones as they migrate through the gel. Field-flowfractionation is carried out in a thin ribbon-like channel, in which theflow profile is parabolic. Particles are loaded as a sample zone, andthen flow through the channel. Separation occurs as particles ofdifferent properties flow in different positions of the flow, due to theinfluence of a field, resulting in different migration speeds. The fieldis applied perpendicular to the flow. Sedimentation utilizesgravitational or centrifugal acceleration to force particles through afluid. Particles migrate through the fluid at different speeds,depending on their sizes and densities, and thus are separated. Sizeexclusion chromatography (SEC) utilizes a tube packed with porous beads,through which sample molecules are washed. Molecules smaller than thepores can enter the beads, which lengthen their migration path, whereasthose larger than the pores can only flow between the beads. In this waysmaller molecules are on average retained longer and thus becomeseparated from larger molecules. Zones broaden, however, as they passthrough the column, because there are many possible migration paths foreach molecule and each path has a different length, and consequently adifferent retention time. This multipath zone broadening (Eddydiffusion) is a major factor limiting resolution. J. C. Giddings,Unified Separation Science (John Wiley & Sons, New York, 1991). Othermethods for separation according to size, including gel electrophoresis,field-flow fractionation, also involve stochastic processes, which maylimit their resolution. J. C. Giddings, Nature 184, 357 (1959); J. C.Giddings, Science 260, 1456 (1993).

The need for reliable and fast separation of large biomolecules such asDNA and proteins cannot be overemphasized. Recently,micro/nano-fabricated structures exploiting various ideas for DNAseparation have been demonstrated. The use of micro/nano-fabricatedstructures as sieving matrices for particle separation was disclosed inU.S. Pat. No. 5,427,663. According to this document, DNA molecules areseparated as they are driven by electric fields through an array ofposts. U.S. Pat. No. 5,427,663 discloses a sorting apparatus and methodfor fractionating and simultaneously viewing individual microstructuresand macromolecules, including nucleic acids and proteins. According toU.S. Pat. No. 5,427,663, a substrate having a shallow receptacle locatedon a side thereof is provided, and an array of obstacles outstandingfrom the floor of the receptacles is provided to interact with themicrostructures and retard the migration thereof. To create migration ofthe microstructures, electrodes for generating electric fields in thefluid are made on two sides of the receptacle. This is analogous to theconventional gel electrophoresis. However, micromachined structures aresubstituted for gel as sieving matrices.

A variety of microfabricated sieving matrices have been disclosed. Inone design, arrays of obstacles sort DNA molecules according to theirdiffusion coefficients using an applied electric field [Chou, C. F. et.al., Proc. Natl. Acad. Sci. 96, 13762 (1999).]. The electric fieldpropels the molecules directly through the gaps between obstacles,wherein each gap is directly below another gap. The obstacles are shapedso that diffusion is biased in one direction as DNA flows through thearray. After flowing through many rows of obstacles, DNA with differentdiffusion coefficients are deflected to different positions. However,because the diffusion coefficient is low for large molecules, theasymmetric obstacle arrays are slow, with running times of typicallymore than 2 hours. In a second design, entropic traps consisting of aseries of many narrow constrictions (<100 nm) separated by wider anddeeper regions (a few microns), reduce the separation time to about 30minutes [Han, J. & Craighead, H. G., Science 288, 1026 (2000).]. Becausethe constrictions are fabricated to be narrower than the radius ofgyration of DNA molecules to be separated, they act as entropicbarriers. The probability of a molecule overcoming the entropic barrieris dependent on molecular weight, and thus DNA molecules migrate in theentropic trap array with different mobilities. Larger molecules, withmore degrees of configurational freedom, migrate faster in thesedevices. In a third design, a hexagonal array of posts acts as thesieving matrix in pulsed-field electrophoresis for separation of DNAmolecules in the 100 kb range [Huang, L. R., Tegenfeldt, J. O., Kraeft,J. J., Sturm, J. C., Austin, R. H. and Cox, E. C., Nat Biotechnol. 20,1048 (2002).]. However, these devices generally require features sizescomparable to or smaller than the molecules being fractionated. Han, J.& Craighead, H. G. Separation of long DNA molecules in a microfabricatedentropic trap array. Science 288, 1026-1029 (2000); Turner, S. W.,Cabodi, M., Craighead, H. G. Confinement-induced entropic recoil ofsingle DNA molecules in a nanofluidic structure. Phys Rev Lett. 2002Mar. 25; 88(12):128103; Huang, L. R., Tegenfeldt, J. O., Kraeft, J. J.,Sturm, J. C., Austin, R. H. and Cox, E. C. A DNA prism for high-speedcontinuous fractionation of large DNA molecules. Nat Biotechnol. 2002October; 20(10):1048-51; and Huang, L. R., Silberzan, P., Tegenfeldt, J.O., Cox, E. C., Sturm, J. C., Austin, R. H. and Craighead, H. Role ofmolecular size in ratchet fractionation. Phys. Rev. Lett. 89, 178301(2002). The need for small feature size may have the followingdetrimental effects: (i) the devices cannot fractionate small moleculessuch as proteins, (ii) the devices may have very low throughput, andthus are not useful sample preparation tools, (iii) the devices can onlyanalyze very small volume of samples, and therefore usually requireconcentrated samples or expensive equipment for sample detection, and(iv) manufacturing the devices require state-of-the-art fabricationtechniques, and thus high cost.

SUMMARY OF THE INVENTION

The present invention provides a microfluidic device for separatingparticles according to size comprising a microfluidic channel, and anarray comprising a network of gaps within the microfluidic channel. Thedevice employs a field that propels the particles being separatedthrough the microfluidic channel. The individual field flux exiting agap is divided unequally into a major flux component and a minor fluxcomponent into subsequent gaps in the array, such that the averagedirection of the major flux components is not parallel to the averagedirection of the field.

In a preferred embodiment, the present invention provides a microfluidicdevice for separating particles according to size comprising amicrofluidic channel, and an ordered array of obstacles within themicrofluidic channel. The ordered array of obstacles is asymmetric withrespect to the average direction of the applied field. The deviceemploys a field that propels the particles being separated through themicrofluidic channel.

The present invention also provides a method for separating particlescomprising introducing the particles to be separated into an arraycomprising a network of gaps within the microfluidic channel andapplying a field to the particles to propel the particles through thearray. A field flux from the gaps is divided unequally into a major fluxcomponent and a minor flux component into subsequent gaps in the array,such that the average direction of the major flux components is notparallel to the average direction of the field.

In a preferred embodiment, the present invention also provides a methodfor separating particles according to size comprising: introducing theparticles to be separated into a microfluidic channel comprising anordered array of obstacles, and applying a field to the particles,wherein the ordered array of obstacles is asymmetric with respect to theaverage direction of the applied field.

In another embodiment, the present invention provides a microfluidicdevice for separating particles according to size comprising amicrofluidic channel, and multiple arrays in series within themicrofluidic channel, wherein each array has a different critical size.The device employs a field that propels the particles being separatedthrough the microfluidic channel. Each of the arrays comprises a networkof gaps wherein a flux of the field from the gaps is divided unequallyinto a major flux component and a minor flux component into subsequentgaps in the network. The average direction of the major flux componentsin each array is not parallel to the average direction of the field.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of an array device according to anembodiment of the present invention. The device consists of an obstaclearray asymmetric about the field direction.

FIG. 2 shows a schematic diagram of a perspective view of a preferredembodiment of the invention, comprising a base substrate with a mainsurface, on which an obstacle course is made to form a network of gaps,wherein cascades of unequal bifurcations of field flux occur. Theobstacle course may be sealed to a cap layer to form an enclosed networkof gaps.

FIG. 3 shows a schematic diagram of an obstacle array. The obstaclearray is symmetric about the dotted lines (principle axis), butasymmetric with respect to the field direction.

FIG. 4 shows a schematic diagram of an embodiment of the invention,comprising a network of gaps defined by an array of obstacles. Eachstreamline represents an equal amount of field flux. The field flux fromone gap is divided into two subsequent gaps, wherein the amounts of fluxgoing into the two gaps are unequal. In this case, two streamlines goesto the left (major flux component) and one streamline goes to the right(minor flux component) at each gap. The array direction is indicated bythe gray arrow.

FIG. 5 shows a schematic diagram of particles separated in an arraydevice according to an embodiment of the present invention. The smallparticles move along field and large ones towards array direction.

FIG. 6 shows a schematic diagram of an obstacle array, showingcharacteristic array dimensions. λ denotes the period of a row ofobstacles, d the gap spacing between obstacles, and aλ the lateral shiftof every row.

FIG. 7 shows a schematic diagram of a particular array, showing thatfield lines go around obstacles. The dotted line at the center marks thedivision of field lines going to different sides of the obstacle.

FIG. 8 shows a schematic diagram of a particular array illustrating thatfield lines shift relative positions in gaps. To illustrate, a=1/3 andeach gap is divided into three slots, which are denoted 1, 2 and 3.Field lines passing through slot 1 go to slot 2 in the next gap, thosethrough slot 2 go to 3, and the ones through 3 to 1.

FIG. 9 shows a schematic diagram of a particular array illustrating thepath of small particles.

FIG. 10 shows a schematic diagram of a particular array illustrating thedisplacement of large particles by obstacles.

FIG. 11 shows a schematic diagram of a particular array illustrating thepath of large particles. Large particles move along the array direction(displacement mode). Note that even if the particle starts at the leftside of the gap, eventually it moves to the right side and stays at theright as it is constantly being displaced by obstacles.

FIG. 12 shows a plot of particle migration direction vs. their size.There exists a critical particle size R₀ where a sharp transition ofmigration direction occurs.

FIG. 13 shows a schematic diagram of two arrays of different criticalsizes in series. Particles at output 1 are smaller than R₁, the criticalsize of the first array in the series, those at output 2 between size R₁and R₂, and the ones at output 3 are larger than R₂, the critical sizeof the second array.

FIG. 14 shows a schematic diagram of multiple arrays in series, whereineach array has a different critical size. The dotted horizontal linerepresents the division between subsequent arrays and each subsequentarray in the series has an increasing critical size. A continuousdistribution of molecular sizes can thus be fractionated and analyzedwith one device.

FIG. 15 shows a schematic top view of an embodiment of the invention,using a square array of circular obstacles to form the network of gaps,tilted at a small angle θ with respect to the field. The square array isspanned by primitive vectors x and y, which are perpendicular to eachother. Bifurcation of field flux occurs because of the asymmetry of theobstacle array with respect to the field.

FIG. 16 shows a schematic top view of an embodiment of the invention.Each obstacle is centered at a lattice point, spanned by the primitivevectors x and y. However, the obstacles may have different shapes.

FIG. 17 shows a schematic top view of an embodiment of the invention.Every obstacle may be identical with the same horizontal period λ.However, every row of obstacles is shifted horizontally by a differentamount with respect to the previous row, and the obstacle course is nota periodic lattice.

FIG. 18 shows a schematic diagram of a microfluidic device forcontinuous-flow separation, with sample loading structures.

FIG. 19 shows a diagram of a microfluidic device for continuous-flowseparation as constructed in Example 1.

FIG. 20 shows fluorescent images of microspheres migrating in theobstacle array, showing (A) the two transport modes for (B) theseparation of microspheres with no dispersion. The gray dots in (A),which represent the obstacles, have been superimposed on the fluorescentimage.

FIG. 21 shows the fluorescent profiles of microspheres separated usingflow speeds of ˜40 μm/s (upper curves) and ˜400 μm/s (lower curves)scanned at ˜11 mm from the injection point. The 0.60 μm, 0.80 μm, and1.03 μm diameter beads are green-fluorescent, while 0.70 μm and 0.90 μmare red, and thus each scan is shown as two curves representing the twocolors.

FIG. 22 shows the measured migration angles as a function of microspherediameter at two different flow speeds.

FIG. 23 shows a schematic diagram of a device for particle separationcomprising an array having 9 sections (divided by dotted lines) ofdifferent critical sizes, and sample loading structures. While theorientation and the lattice constants of the array are kept the same,the obstacle diameters are changed to create different-sized gaps d andcritical sizes.

FIG. 24 shows the high-resolution separation of fluorescent microspheresof 0.80 μm (left), 0.90 μm (center) and 1.03 μm (right), using an arrayof varying gap size. While the orientation and the lattice constants ofthe array are kept the same, the obstacle diameters are changed tocreate different-sized gaps d, labeled on the left side of thefluorescent image. Individual 1.03 μm streamlines clearly show zigzagmigration.

FIG. 25 shows the fluorescent profile of the separated particles scannedat 14 mm from the injection point.

FIG. 26 shows of fluorescent image of DNA molecules separated accordingto size using a device of the present invention. Molecules areintroduced at the top of the figure.

FIG. 27 shows a schematic diagram of a device used to concentratesample. The field direction can be defined by the sidewalls of thearray. The shaded area shows the trajectories of particles larger thanthe critical size, injected from the top. Particles move against thesidewall and are concentrated.

FIG. 28 shows a schematic diagram of a device for concentratingparticles. The device comprises an array, which moves particles againsta boundary of the array, and microfluidic channels and reservoirs forsample loading and collection.

FIG. 29 shows of fluorescent image of DNA molecules getting concentratedagainst a boundary of the array. DNA samples were introduced from thetop of the picture.

FIG. 30 shows a schematic diagram of a device having an ordered array ofobstacles and employing a non-uniform field, wherein the direction ofthe field changes across the array.

FIG. 31 shows a schematic diagram of a device having an ordered array ofobstacles in a curved microfluidic channel. The curved channel mayresult in a field that is non-uniform.

DETAILED DESCRIPTION

The present invention provides a method and a device for separatingparticles according to size using an array comprising a network of gaps,wherein the field flux from a gap divides unequally into subsequentgaps. A field is applied to the array to propel the particles beingseparated through the array. In a preferred embodiment, the array is anordered array of obstacles in a channel, wherein the array is asymmetricwith respect to the direction of the applied field. In further preferredembodiments, the channel that contains the array is a microfluidicchannel. The term “channel” as used herein refers to a structure whereinfluid may flow. A channel may be a capillary, a conduit, a strip ofhydrophilic pattern on an otherwise hydrophobic surface wherein aqueousfluids are confined, etc. The term “microfluidic” as used herein, refersto a system or device having one or more fluidic channels, conduits orchambers that are generally fabricated at the millimeter to nanometerscale, e.g., typically having at least one cross-sectional dimension inthe range of from about 10 nm to about 1 mm.

The present invention is useful for the separation of biologicalparticles according to size, including bacteria, cells, organelles,viruses, nucleic acids (i.e., DNA, etc.), proteins and proteincomplexes, as well as other nonbiological particles suspended in fluid,such as industrial polymers, powders, latexes, emulsions, and colloids.In addition to particle separation, the method can be used to analyzethe size distribution of samples, or extract particles of certain sizerange from mixtures of particles. Further, because of the large featuressize, the device may be a high throughput sample preparation tool. Thepresent invention may provide the advantages of low manufacturing cost,high resolution, and improved sample throughput.

In a preferred embodiment, the present invention provides a microfluidicdevice that separates particles in fluid according to size. A field isapplied to the particles being separated as the particles pass throughthe array. The term “field” as used herein refers to any force or vectorentity that can be used to propel the particles through the array. Thefield that drives the particles being separated may be a force field,such as an electric field, a centrifugal field, or a gravitationalfield. The field that drives the particles being separated may also be afluid flow, such as a pressure-driven fluid flow, an electro-osmoticflow, capillary action, etc., wherein the vector entity is the fluidflux density. Further, the field may be a combination of a force fieldand a fluid flow, such as an electro-kinetic flow, which is acombination of the electric field and the electro-osmotic flow. Inpreferred embodiments, the average direction of the field will beparallel to the walls of the channel that contains the array.

The array for use in the present invention comprises a network of gapsthat creates a field pattern such that the field flux from a gap withinthe network is divided into unequal amounts (a major flux component anda minor flux component) into the subsequent gaps (for example, see FIG.4), even though the gaps may be identical in dimensions. Looking at thearray as a whole, on average the unequal divisions of the field flux isweighted in one direction. Thus, the major flux components are diverted,on average, in the same direction (i.e., diverted to the same side ofthe obstacle), such that the average direction of the major fluxcomponents is not parallel to the average direction of the minor fluxcomponents, or to the average direction of the field. It is preferredthat a majority of the major flux components be diverted in the samedirection. In a particularly preferred embodiment, the array is anordered array, wherein the major flux component from each bifurcationevent is diverted in the same direction. Generally, the minor fluxcomponent exiting one gap feeds into the major flux component exiting asubsequent gap (FIG. 4).

The term “gap” as used herein refers to a structure wherein fluids orparticles may flow. A gap may be a capillary, a space between twoobstacles wherein fluids may flow, a hydrophilic pattern on an otherwisehydrophobic surface wherein aqueous fluids are confined, etc. In apreferred embodiment of the invention, the network of gaps is defined byan array of obstacles. In this embodiment, the gaps would be the spacebetween adjacent obstacles. In a preferred embodiment, the network ofgaps will be constructed with an array of obstacles on surface of asubstrate (FIG. 2). The term “obstacle” as used herein refers to aphysical structure or pattern wherein the particles being separatedcannot penetrate, and thus an obstacle may refer to a post outstandingon a base substrate, a hydrophobic barrier for aqueous fluids, etc. Insome embodiments, the obstacle may be permeable to the field. Forexample, an obstacle may be a post made of porous material, wherein thepores allows penetration of the field, but are too small for theparticles being separated to enter.

In a preferred embodiment, the network of gaps, wherein the field fluxis divided unequally, is formed using a periodic array of obstacles,which is asymmetric with respect to the average direction of the field.An important feature of this embodiment is that the obstacle array isasymmetric with respect to the field, even though the array itself maybe symmetric with respect to other axes (FIG. 3). The term asymmetric asused herein refers to an obstacle array in which the field flux from agap between two obstacles is bifurcated by a subsequent obstacle, suchthat the bifurcated field flux is not divided into two equal amounts(for example, see the field stream lines of FIG. 4). This may beachieved when the average direction of the field is not parallel to aprinciple axis of the array (FIG. 3). In one embodiment, an orderedarray is tilted at an offset angle θ with respect to the field (forexample, FIG. 15), wherein the offset angle θ is selected such that thearray is not aligned to the field (i.e., is asymmetric to the field).Alternatively, an asymmetric array may also be achieved using an arraycomprising rows of obstacles in which each row is laterally shifted fromthe previous row and the misalignment factor, a, is larger than 0 andsmaller than 0.5 (FIG. 6). In a preferred embodiment, the array ofobstacles will be an ordered array in which a principle axis of thearray is not parallel to the direction of the field (FIG. 3). The termasymmetric refers to the array of obstacles (for example, to the arrayaxis) and not to the shape of individual obstacles. It is preferred thatthe array is an ordered array in order to maximize the number ofbifurcation events that can occur as the sample passes through the array(for example, see FIG. 4).

As used herein, the term “ordered” refers to an array having a generallyperiodic or repeating spatial arrangement. For example the repeatingspatial arrangement, may be square, rectangular, hexagonal, oblique,etc. In other embodiments, the array need not be an ordered array.

In one embodiment of the invention, particles flow through an asymmetricobstacle array, and are separated according to size into differentstreams (FIG. 5). While small particles follow the field direction,large particles migrate at the array direction. The array directioncorresponds to the average direction of the major component of the fieldflux (for example, see the gray arrow of FIG. 4). The basic theory ofthe transport process is schematically illustrated in FIG. 5. FIG. 5depicts one embodiment of an asymmetric array of obstacles in which thearray is misaligned (i.e., asymmetric) to the field. For the array typedepicted in FIG. 6, the misalignment factor, a, is larger than 0 andsmaller than 0.5. When a=0 or a=0.5, the array is symmetric about thefield axis. As shown in FIG. 6, each row of obstacles is shiftedhorizontally with respect to the previous row by aλ, where λ is thecenter-to-center distance between the obstacles. For the purpose of thisdiscussion, let us assume that a equals 1/3. In preferred embodiments,particles to be separated are driven through the array by a fluid flow.Because of the low Reynolds number in these devices, the flow lines maybe laminar, resulting in negligible turbulence and inertial effects.

Because the obstacle array is asymmetrically aligned to the field, i.e.,the obstacle lattice is asymmetric with respect to the average fielddirection, field lines going through one gap have to go around theobstacle in the next row (FIG. 7). To consider the allocation of fieldlines in the next row, we assume that the field in the gap betweenobstacles is locally uniform, i.e. field lines are equally spaced inevery gap. This may be a good approximation if the field is electrical.This assumption may be relaxed in the case of other fields. The totalfield flux Φ going through one gap is divided by the subsequent obstacleand splits into two streams, the major flux component and the minor fluxcomponent, as the flux goes into the two subsequent gaps (FIG. 7). Ifthat the average field direction is vertical (parallel to the channelwalls), aΦ has to go to one gap and (1-a)Φ to the other. Therefore,unequal division of the field flux occurs at each gap. Generally, theminor flux component exiting one gap feeds into the major flux componentexiting a subsequent gap (FIG. 4).

If we divide each gap into 1/a slots (FIG. 8, to illustrate a=1/3), eachfield line shifts to the next slot as it passes through a row in acyclic manner: field lines going through position 1 will be at position2 in the next row, those at position 2 will go to position 3 . . . theones at position 1/a will go back to position 1. Small particles followthe field and shift from one position to the next in a cyclic manner(FIG. 9). Because small particles may go back to their original slot(relative position in the gap) after 1/a rows, their trajectories followthe field direction and do not disperse after going around manyobstacles. Because of the “zigzag” motion of the small particles, thistransport pattern may be referred to as the “zigzag mode.”

In contrast, particles with a large diameter compared to the slot widthwill not follow individual streamlines, but instead be propelled by manystreamlines, which fundamentally changes their final direction ofmigration. As shown in FIG. 10, a particle in contact with an obstacleand whose characteristic radius R is larger than ad (the width of thegap), will follow a field line going through slot 2, because its centerfalls in slot 2. Now the field lines goes to slot 3 in the next gap, sothe center of the particle should also be in slot 3. However, slot 3does not have enough room for the particle. Therefore the particle isdisplaced back to slot 2. This process may be repeated every time as alarge particle approaches a row of obstacles. Because large particlesare displaced from slot 3 to slot 2, they follow the array direction(FIG. 11). The motion of large particles through the array is termed the“displacement” mode.

In summary, there exists a critical particle radius R₀ above whichparticles move in the array direction (displacement mode), and smallerthan which particles follow the average field direction (zigzag mode)(FIG. 12). The critical size, R₀, may be determined by a and d, where ais the misalignment factor and d is the gap width. In the case wherefields are evenly distributed in gaps, R₀=ad. Thus, a particle'shydrodynamic radius (size) determines which transport mode it follows.Also note that the above theory can be easily generalized for otherfields, such as electrophoretic fields or pressure driven fluid flows byconsidering ion flows instead of fluid flows.

In one embodiment of the invention, the device may be employed as afilter. The term “filter” as used herein refers to a device whichremoves particles in certain size ranges from a fluid. The sharptransition of migration angle with respect to size is ideal for filters(FIG. 12). The micro/nano-structures of the arrays are preferably madeby micro/nano-fabrication techniques. FIG. 13 shows the schematicdiagram of a filter of the invention, wherein injected particles areseparated into three groups of different sizes. In principle, one arraycan set up one critical radius (size) for particle separation, andseparate a mixture of particles into two groups of particles, in one ofwhich particles are larger than the critical size, and in the other ofwhich particles are smaller. Two arrays of different critical sizes puttogether in series can separate a mixture of particles into three sizeranges, large, medium, and small (FIG. 13). The sample of particles ofdifferent sizes is injected in the first array, which has a smallercritical size (R₁) than the second (R₂). While the large andmedium-sized particles move in the displacement mode in the first array,the small ones move in the zigzag mode and are separated out. Asparticles move into the second array, the medium-sized particles switchto the zigzag mode and are separated out from the large ones. One cancollect molecules of different sizes at the output of the second array,which may serve as a molecular filter and a sample preparation tool.Further, the size range of particles going to output 2, determined by R₁and R₂, can be very narrow. For example, the device can be designed tosingle out only one size of DNA restriction fragments from tens orhundreds of other sizes of fragments. This device could replace theconventional techniques of gel electrophoresis and gel cutting.

In another embodiment of the invention, the device of the invention mayfractionate molecules according to size. High selectivity can beachieved at the sharp transition region (FIG. 12), using a single arrayof fixed critical radius (size). Alternatively, separation of particlesin a broad size-range may require lower selectivity, which could beachieved using many arrays in series, each of which has a differentcritical size (FIG. 14). FIG. 14 shows a schematic diagram of multiplearrays in series, wherein each array has a different critical size. Thedotted horizontal line represents the division between subsequent arraysand each subsequent array in the series has a increasing critical size.A continuous distribution of molecular sizes can thus be fractionatedand analyzed with one device. In fact, smoothmigration-angle-to-particle-size characteristics, including linear andexponential relationships, can be designed using many arrays in series,each having a different critical size. The critical size of a array maybe adjusted by changing the gap width d, by shifting obstacles, or both.

In one embodiment of the invention, the device may comprise an orderedarray of obstacles and may employ a non-uniform field, wherein thedirection of the field changes across the array (FIG. 30). In oneembodiment, the changing field directions may be created by a curvedmicrofluidic channel. Since the critical size of the array depends onthe field direction, the non-uniform field creates sections of array ofdifferent critical sizes. This may be desired for fractionation ofparticles in a broad size range. In the particular embodiment shown inFIG. 30, the field direction is large at the first array section andthen reduced at the following array sections, creating a large criticalsize in the first section and smaller critical sizes at the followingsections.

In another embodiment of the invention, the device comprises an orderedsquare array of obstacles in a curved microfluidic channel (FIG. 31).The curved channel may result in a field that is non-uniform. The changein the field direction across the array creates a gradient of criticalsize in the array, because the critical size may depend on the fielddirection. Further, the field strength, another parameter that can betuned, may be adjusted by changing the width of the microfluidicchannel. This device may have better separation range and resolutionthan an array of one fixed critical size. A continuous gradient ofcritical size across the array can also be created by changing gapwidths.

In one embodiment of the invention, the array comprises a square latticeof cylindrical obstacles, with the array tilted at an offset angle θwith respect to the field (FIG. 15). An offset angle θ of tan⁻¹ (a) isequivalent to a misalignment factor of a in FIG. 6.

In another embodiment of the invention, the array comprising a networkof gaps may be formed by a course of obstacles, which are not identical(FIG. 16). More specifically, obstacles may have different shapes ordimensions. Because the lattice on which obstacles overlay is asymmetricwith respect to the field, the field flux in each gap between obstaclesis divided unequally into the subsequent gaps.

In another embodiment of the invention, the obstacle course, which formsthe network of gaps, may not be a periodic lattice. For example, FIG. 17shows a schematic top view of an embodiment of the invention in whicheach obstacle may be identical and have the same horizontal period λ.However, every row of obstacles is shifted horizontally by a differentamount with respect to the previous row, and the obstacle course is nota Bravais lattice [N. W. Ashcroft and N. D. Mermin, Solid State Physics(Saunders College Publishing, 1976)]. Nonetheless, field flux in thearray undergoes cascades of bifurcations because the obstacle course isasymmetric with respect to the average field direction. Because thecritical size is dependent on the asymmetry of the field flux division,which originated from the asymmetry of the array with respect to thefield, this embodiment may be useful for separating particles in a broadrange.

In another embodiment of the invention, particles are loaded into thearray using many microfluidic channels at the top of the array (FIG.18). The many channels generate a field pattern that is generallyuniform across the array. Particles are injected from one or morechannels connected to one or more reservoirs containing the particles,which are to be carried across the array by the field.

In another embodiment of the invention, particles are unloaded from thearray, for example via microfluidic channels at the end of the array(FIG. 18). The particles can then be routed to the next component of themicrofluidic chip for further use.

In another embodiment, the device of the invention can concentrateparticles larger than a critical size (FIG. 27). This embodiment mayexploit the array's ability to move particles off the applied fielddirection in displacement mode. Particles are introduced on one side ofthe array. Particles larger than the critical size of the array (movingin displacement mode) get piled against another boundary of the array.The device of the invention in this case acts as a concentrator. Theconcentrated sample can then be routed to the next component of themicrofluidic chip for further use, such as creating a sample zone forfractionation. The concentrated sample can also be taken off the chipfor further applications.

In a preferred embodiment of the invention, the device ismicro/nano-fabricated. Microfabrication techniques may be selected fromthose known in the art, for example, techniques conventionally used forsilicon-based integrated circuit fabrication, embossing, casting,injection molding, and so on [E. W. Becker et. al., MicroelectronicEngineering 4 (1986), pages 35 to 56]. Examples of suitable fabricationtechniques include photolithography, electron beam lithography, imprintlithography, reactive ion etching, wet etch, laser ablation, embossing,casting, injection molding, and other techniques [H. Becker et. al., J.Micromech. Microeng. 8 (1998), pages 24 to 28]. The microfluidic devicemay be fabricated from materials that are compatible with the conditionspresent in the particular application of interest. Such conditionsinclude, but are not limited to, pH, temperature, application of organicsolvents, ionic strength, pressure, application of electric fields,surface charge, sticking properties, surface treatment, surfacefunctionalization, and bio-compatibility. The materials of the deviceare also chosen for their optical properties, mechanical properties, andfor their inertness to components of the application to be carried outin the device. Such materials include, but are not limited to, glass,fused silica, silicone rubber, silicon, ceramics, and polymericsubstrates, e.g., plastics, depending on the intended application.

It is understood that the various embodiments described herein are byway of example only, and are not intended to limit the scope of theinvention. For example, many of the materials and structures describedherein may be substituted with other materials and structures withoutdeviating from the spirit of the invention. It is understood thatvarious theories as to why the invention works are not intended to belimiting.

EXAMPLES

Specific representative embodiments of the invention will now bedescribed, including how such embodiments may be made. It is understoodthat the specific methods, materials, conditions, process parameters,apparatus, and the like do not necessarily limit the scope of theinvention.

Example 1

A microfluidic device was constructed (FIG. 19). The microfluidicchannel is 16 mm long, 3.2 mm wide, and 10 μm deep. The array fillingthe channel consists of a square lattice of cylindrical obstacles (FIG.19A), where the center-to-center distance, λ is 8 μm, and the spacing dbetween the obstacles 1.6 μm (FIG. 19B). The field employed here is apressure driven field flow. The lattice is rotated by 5.7° (tan⁻¹ 0.1)with respect to the channel (FIG. 19A), which defines the flowdirection. The rotation of tan⁻¹ 0.1 corresponds to a=0.1 thus acomplete pitch λ is shifted every 10 rows (FIG. 19A). In principle, thisconfiguration provides 10 slots, rather than the 3 discussed above.Particles are injected from a 10 μm-wide channel and carried across thearray by a pressure-driven flow, which is made uniform by the manychannels on the top and bottom of the array (FIG. 19C). The microfluidicchannels and the array were fabricated on a silicon wafer usingphotolithography and deep reactive ion etching, techniquesconventionally used for silicon-based integrated circuit fabrication.Holes through wafers for fluid access were drilled before sealing with aglass coverslip coated with silicone rubber (RTV-615 from GeneralElectric) to sandwich the array.

The two transport modes were experimentally observed using fluorescentpolystyrene microspheres of 0.40 μm and 1.03 μm in aqueous buffer (FIG.20A) (0.1× Tris-Borate-EDTA buffer containing 0.02% POP-6, aperformance-optimized linear polyacrylamide (Perkin-Elmer Biosystems),was used in the experiments.). The image was taken by fluorescentmicroscopy with a long exposure time to show trajectories of individualparticles. The varying brightness along the trajectory reflects thedifferent flow speeds of a microsphere in the array, dimmer in thenarrow gaps due to higher flow speed and lower residence time. Aspredicted, the 0.40 μm microsphere (left) crossed a column of obstaclesevery 10 rows in the zigzag mode, whereas the 1.03 μm microsphere(right) was channeled along the axis of the obstacle array in thedisplacement mode.

FIG. 20B shows the continuous-flow separation of 0.40 μm and 1.03 μmmicrospheres injected into the array from a feed channel at the top. Inthis image many trajectories were superimposed. The 0.40 μm and 1.03 μmspheres migrated at ˜0° (flow direction) and ˜5.7° (array rotation)respectively relative to the flow direction, as expected. The pressureused to drive the flow was 30 kPa, which created an average flow speedof ˜400 μm/s. The running time through the device was ˜40 s.

To probe the resolution of the device, fluorescent microspheres of 0.60μm, 0.70 μm, 0.80 μm, 0.90 μm and 1.03 μm diameter were mixed andinjected into the array (The concentrations of the microspheres of 0.60μm, 0.70 μm, 0.80 μm, 0.90 μm and 1.03 μm were 0.015%, 0.010%, 0.010%,0.005%, and 0.005% solid, respectively). The beads were separated intodifferent streams using a flow speed of ˜40 μm/s, created by a drivingpressure of 3 kPa. The fluorescence profile scanned 11 mm from theinjection point is shown in FIG. 21. The measured migration directionswith respect to the flow (field), defined as the migration angles, areplotted as a function of the microsphere size in FIG. 22, which showsthat under this flow speed (˜40 μm/s), the transition from the zigzag tothe displacement mode is gradual. The smooth transition is probably dueto Brownian motion of the particles between streamlines. At a flow speedof 40 μm/s, a 0.6 μm particle—diffusion coefficient in water of 0.73μm²/s (H. C. Berg, Random Walks in Biology, Princeton University Press,New Jersey, 1993, p. 56)—has a diffusion length of ˜0.54 μm over the 0.2s it takes to move the 8 μm from one row of obstacles to the next. Thisis a factor of 3 larger than the average slot width of ˜0.16 μm.

To minimize the effects of Brownian motion, the Peclet number wasincreased by increasing the flow speed. Peclet number (Pe) is defined as${{Pe} = \frac{vd}{D}},$where v is the flow speed, d is the characteristic dimension of thearray, and D the diffusion coefficient of the particle being separated.FIG. 22 shows a sharper transition when the flow speed is increased by afactor of 10 (˜400 μm/s), created by a driving pressure of 30 kPa. Thehigh flow speed not only increases the selectivity so that the devicebecomes more sensitive to size changes, but also shortens the runningtime to ˜40 s. The transition occurs approximately at 0.8 μm (FIG. 22).The size coefficients of variation (CV) of the best monodispersedmicrospheres commercially available are 1.3%, 1.0% and 1.0% for 0.70 μm,0.80 μm and 0.90 μm, respectively, as measured by the manufacturer. Thecoefficient of variation (CV) of particle diameter φ is defined as${{CV} = {\frac{\Delta\phi}{< \phi >} \times 100\%}},$where Δφ is the standard deviation of φ, and <φ> the mean of φ. The peakwidths measured at 11 mm from the injection point, correspond to CV's of2.5%, 1.2% and 0.9% for these three sizes, respectively (FIG. 21). CV'sare calculated according to:${{CV} = {\frac{\frac{\mathbb{d}\phi}{\mathbb{d}x}\Delta\quad x}{< \phi >} \times 100\%}},$where x is the lateral position of the band and Δx the measured standarddeviation (half-width). Note that the measured peaks of 0.80 μm and 0.90μm are as sharp as the size-variances of the microspheres themselves,and thus band broadening in our device is less than the known variancein particle size.

Example 2

One advantage of the flexibility of microfabrication is that the arraycan be designed to have varying gap widths as a function of distance,thereby optimizing separation for complex mixtures. To demonstrate thispoint, a device was fabricated containing 9 sections, each of which hada different gap width, starting with 1.4 μm and ending with 2.2 μm inincrements of 0.1 μm (FIGS. 23 and 24). The varying gap widths weredesigned to tune the critical diameter in 9 stages from ˜0.70 μm to˜1.10 μm, so that a given sized particle would switch from displacementmode to zigzag mode as the gap width increased.

A mixture of monodisperse (CV=1%) microspheres of 0.80 μm, 0.90 μm and1.03 μm was injected from the small-gap side of the array (FIGS. 23 and24), and flown at ˜400 μm/s using a driving pressure of 30 kPa.Initially, all microspheres were larger than the critical size, andmigrated at the same angle with respect to the vertical flow(displacement mode). Soon, however, the 0.80 μm microspheres (left)switched to the flow direction (vertical), presumably in zigzag mode.0.90 μm microspheres switched to vertical at the fourth section, and the1.03 μm microspheres made the transition around the eighth section. Thefluorescent intensity profile was scanned at ˜14 mm from the injectionpoint (FIG. 25), and showed that the 0.80 μm, 0.90 μm and 1.03 μm peakshad CV's of 1.1%, 1.2% and 1.9%, respectively (see the scale bars,centered at the means of the peaks). By comparison, the 1% CVattributable to nonuniformity in the microsphere population is shown asthe black scale bars underneath the peaks. Note that a fraction of the1.03 μm microspheres were separated out from the main peak and formed asub-band, most likely because of the non-homogeneity in the microspherepopulation. Again, the peaks are virtually resolved to themono-dispersity of the most uniform microspheres commercially available.The running time was ˜40 s.

The results of FIGS. 21 and 25 suggest that the resolution of the devicemay exceed our ability to measure it. One could in principle discoverthe limits of resolution for the approach by exploring the resolvingpower of the current array with a series of size classes varying inapproximately 0.01 μm intervals around ˜1 μm, each class with a CV of˜0.1%.

Example 3

A device having identical array dimensions and loading structures as inExample 1 was constructed for separation of nucleic acids according tomolecular weight. The device was made of fused silica instead of siliconusing the same techniques as described in Example 1. Electric fields,instead of pressure-driven fluid flow, were used as the field. A mixtureof coliphage λ and T2 dsDNA (48.5 kb and 164 kb respectively) at ˜2μg/ml and ˜1 μg/ml was used as a test sample, and visualized byfluorescent microscopy. DNA was stained with TOTO-1 (Molecular Probes)at a ratio of 1 dye molecule per 10 base pairs. 0.1% POP-6, aperformance-optimized linear polyacrylamide (Perkin-Elmer Biosystems),and 10 mM dithiothreitol (DTT) were added to the 1/2× Tris-Borate-EDTAbuffer to suppress electro-osmotic flow and photo-bleaching,respectively. The electric fields were applied using electrodes immersedin buffer reservoirs. The migration speeds of the molecules werecontrolled by the voltage applied to the reservoirs, and measured byobserving the velocity of individual molecules.

The two species were separated with good resolution using an electricfield of ˜5 V/cm (FIG. 26).

Example 4

A device of the present intervention for concentrating DNA molecules ismade and schematically shown in FIG. 28. The device comprises an arrayand microfluidic channels for sample injection and extraction, which areidentical in dimensions with Example 1. The field used herein is anelectric field, applied using electrodes immersed in the buffer fluidreservoirs. The device is made of fused silica using techniquesdescribed in Example 1. Coliphage T2 DNA at ˜1 μg/ml in 1/2×Tris-Borate-EDTA buffer containing 0.1% POP-6, a performance-optimizedlinear polyacrylamide (Perkin-Elmer Biosystems) and 10 mM dithiothreitol(DTT), was used as a test sample. The DNA was loaded onto the device atthe sample reservoir, and concentrated against a boundary of the arrayto ˜120 μg/ml (FIG. 29). The electric field strength was ˜5 V/cm. Theconcentrated sample is removed from the array and collected in thesample-collection reservoir (FIG. 28).

While the present invention is described with respect to particularexamples and preferred embodiments, it is understood that the presentinvention is not limited to these examples and embodiments. The presentinvention as claimed therefore includes variations from the particularexamples and preferred embodiments described herein, as will be apparentto one of skill in the art.

1-44. (canceled)
 45. A microfluidic device for concentrating particles,having at least a predetermined critical size, the device comprising: amicrofluidic channel, an ordered array of obstacles within themicrofluidic channel, and a boundary, wherein the device employs a fieldthat propels particles through the microfluidic channel; and the orderedarray of obstacles is asymmetric with respect to the average directionof the field, such that, when particles are introduced into the array,particles having a size less than a predetermined critical size aretransported in a first direction, and particles having a size at leastthat of the critical size are transported in a second direction to theboundary, wherein the first and second directions are different, therebyconcentrating the particles having a size at least that of the criticalsize.
 46. The microfluidic device of claim 45, wherein the ordered arrayof obstacles comprises obstacles arranged in rows, wherein eachsubsequent row of obstacles is shifted laterally with respect to theprevious row.
 47. The microfluidic device of claim 45, wherein theordered array of obstacles is tilted at an offset angle θ with respectto the direction of the field.
 48. The microfluidic device of claim 45,wherein the field is fluid flow, electrical, electrophoretic,electro-osmotic, centrifugal, gravitational, hydrodynamic, pressuregradient, or capillary action.
 49. The microfluidic device of claim 48,wherein the field is a fluid flow.
 50. The microfluidic device of claim48, wherein the field is an electrical field.
 51. The microfluidicdevice of claim 45, wherein the particles are bacteria, cells,organelles, viruses, nucleic acids, proteins, protein complexes,polymers, powders, latexes, emulsions, or colloids.
 52. The microfluidicdevice of claim 51, wherein the particles are DNA molecules.
 53. Amethod for concentrating particles, having at least a predeterminedcritical size, the method comprising: introducing particles into amicrofluidic channel comprising a network of gaps within themicrofluidic channel and a boundary; and applying a field to theparticles to propel the particles through the microfluidic channel,wherein a flux of the field from the gaps is divided unequally into amajor flux component and a minor flux component into subsequent gaps inthe network, such that the average direction of the major flux componentis not parallel to the average direction of the field, and particleshaving a size less than a predetermined critical size are transportedgenerally in the average direction of the field, and particles having asize at least that of the critical size are transported generally in theaverage direction of the major flux component to the boundary, therebyconcentrating the particles having a size at least that of the criticalsize.
 54. The method of claim 53, wherein the network of gaps isconstructed from an array of obstacles.
 55. The method of claim 54,wherein the array of obstacles is an ordered array of obstacles.
 56. Themethod of claim 55, wherein the ordered array of obstacles comprisesobstacles arranged in rows, wherein each subsequent row of obstacles isshifted laterally with respect to the.previous row.
 57. The method ofclaim 55, wherein the ordered array of obstacles is tilted at an offsetangle θ with respect to the direction of the field.
 58. The method ofclaim 53, wherein the field is fluid flow, electrical, electrophoretic,electro-osmotic, centrifugal, gravitational, hydrodynamic, pressuregradient, or capillary action.
 59. The method of claim 58, wherein thefield is a fluid flow.
 60. The method of claim 58, wherein the field isan electrical field.
 61. The method of claim 53, wherein the particlesare bacteria, cells, organelles, viruses, nucleic acids, proteins,protein complexes, polymers, powders, latexes, emulsions, or colloids.62. The method of claim 61, wherein the particles are DNA molecules. 63.A method for concentrating particles, having at least a predeterminedcritical size, the method comprising: introducing particles into amicrofluidic channel, comprising an ordered array of obstacles and aboundary; and applying a field to the particles to propel the particlesthrough the microfluidic channel, wherein the ordered array of obstaclesis asymmetric with respect to the average direction of the field, suchthat particles having a size less than a predetermined critical size aretransported in a first direction, and particles having a size at leastthat of the critical size are transported in a second direction to theboundary, wherein the first and second directions are different, therebyconcentrating the particles having at least the predetermined criticalsize.
 64. The method of claim 63, wherein the ordered array of obstaclescomprises obstacles arranged in rows, wherein each subsequent row ofobstacles is shifted laterally with respect to the previous row.
 65. Themethod of claim 63, wherein the ordered array of obstacles is tilted atan offset angle θ with respect to the direction of the field.
 66. Themethod of claim 63, wherein the field is fluid flow, electrical,electrophoretic, electro-osmotic, centrifugal, gravitational,hydrodynamic, pressure gradient, or capillary action.
 67. The method ofclaim 66, wherein the field is a fluid flow.
 68. The method of claim 66,wherein the field is an electrical field.
 69. The method of claim 63,wherein the particles are bacteria, cells, organelles, viruses, nucleicacids, proteins, protein complexes, polymers, powders, latexes,emulsions, or colloids.